MicroRNAs are a class of endogenous small RNAs that are bound by Argonaute proteins to form the microRNA-Induced Silencing Complex (miRISC). MicroRNAs in miRISC negatively regulate complementary protein-coding genes to control many aspects of normal development. The biogenesis of microRNAs and miRISC is extensively studied, but comparatively little is understood about their turnover. Untemplated nucleotide additions at the 3' end of microRNAs are correlated with destabilization, but their functional role remains unclear. The objective of this work is to elucidate the mechanisms of microRNA and miRISC turnover, and to examine the role of 3' nucleotide additions in these processes. First, I will examine the function of candidate genes in 3' nucleotide additions and microRNA decay in the context of acute inactivation of microRNA biogenesis. Second, I will establish a system to measure the turnover of endogenously-expressed Argonaute, and use this system to assess the extent to which microRNA and miRISC turnover are coupled or independent. Third, I will conduct forward genetic screens to identify factors that impact the rate of microRNA turnover. Together, these studies will deepen our understanding of how the abundance of microRNAs and miRISC is regulated, and open up multiple new directions of research. I am uniquely qualified to conduct this research, having studied different aspects of microRNA regulation and biology throughout my graduate and postdoctoral training. In Victor Ambros's laboratory, I have used the C. elegans model system to discover a novel mechanism of post-transcriptional regulation of microRNA activity. In the proposed work, I will apply similar techniques to dissecting microRNA and miRISC turnover, while also taking advantage of the unique focus on RNA biology at the University of Massachusetts Medical School to expand my skill set through collaboration with my advisory committee. My short term career goals are to learn new techniques in biochemistry and cell biology through formal coursework and practical training, and to carry out the proposed research while transitioning to an independent position. My long term goal is to build a multi-faceted research program on the foundation of these projects in a tenure-track position at an academic institution.